Chromosome instability is a hallmark of cancer cells. Balanced translocations, in which arms of nonhomologous chromosomes are joined to one another at specific breakpoints, are distinctive characteristics of certain malignancies. While much has been learned by examining the genes present at translocation breakpoints, almost nothing is known about the molecular mechanisms involved in the genesis of chromosomal translocations. Translocations appear to represent inappropriate double-strand break (DSB) repair events. They are associated with genetic disorders of DNA repair (e.g. Bloom s syndrome), environmental conditions which increase chromosomal breakage (e.g. ionizing radiation), drugs which target enzymes that break and rejoin DNA (e.g. topoisomerase II inhibitors) as well as with cell types that undergo programmed DNA breakage and rejoining (e.g. V(D)J recombination in lymphoid cells). We have developed a simple genetic assay in the model eukaryote Saccharomyces cerevisiae which can easily detect chromosomal rearrangements resulting from nonhomologous recombinational events. In this assay, a double strand break is induced in a marker gene and repair of the break by chromosomal rearrangements is detected by a change in marker gene expression. This assay can be used to study the frequency and spectrum of rearrangements and the system can be modified to accommodate changes in genetic background, changes in the source of DSB, changes in potential target sequences for rearrangements, or changes in the environment in which the cells are growing. The specific aims of this proposal are to 1)examine the in vivo role of topoisomerases in the genesis of chromosomal translocations; 2) determine how drugs that target topoisomerases influence translocation formation; and 3) analyze, in yeast, a segment of human DNA strongly linked to cancer chemotherapy associated translocations. Our long-term objectives are to understand the genetic and environmental factors that predispose chromosomes to translocate and the molecular mechanisms by which specific translocations are generated. Accomplishment of these goals will have great implications for cancer prevention, identification of at-risk individuals, and rational design of chemotherapeutic drugs. The purpose of this exploratory/developmental (R21) proposal is to develop the potential applications of this novel assay and generate sufficient preliminary data to qualify for future research project (i.e. R01) funding in the future.